A defined xeno-free and feeder-free culture system for the derivation, expansion and direct differentiation of transgene-free patient-specific induced pluripotent stem cells

• Published in: Biomaterials 2014-03, Vol.35 (9), p.2816-2826
• Main Authors: Lu, Hong Fang, Chai, Chou, Lim, Tze Chiun, Leong, Meng Fatt, Lim, Jia Kai, Gao, Shujun, Lim, Kah Leong, Wan, Andrew C.A
• Format: Article
• Language: English
• Subjects: Adult; Advanced Basic Science; Animals; Cell Culture Techniques - methods; Cell Differentiation; Cell Proliferation; Cellular Reprogramming; Dentistry; Dopaminergic Neurons - cytology; Dopaminergic Neurons - metabolism; Feeder Cells - cytology; Fibroblasts - cytology; Genetic Vectors - metabolism; Humans; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - metabolism; Lentivirus - metabolism; Mice; Mice, SCID; Parkinson Disease - pathology; Transgenes
• ISSN: 0142-9612; 1878-5905
• DOI: 10.1016/j.biomaterials.2013.12.050

Abstract A defined xeno-free system for patient-specific iPSC derivation and differentiation is required for translation to clinical applications. However, standard somatic cell reprogramming protocols rely on using MEFs and xenogeneic medium, imposing a significant obstacle to clinical translation. Here, we describe a well-defined culture system based on xeno-free media and LN521 substrate which supported i) efficient reprogramming of normal or diseased skin fibroblasts from human of different ages into hiPSCs with a 15–30 fold increase in efficiency over conventional viral vector-based method; ii) long-term self-renewal of hiPSCs; and iii) direct hiPSC lineage-specific differentiation. Using an excisable polycistronic vector and optimized culture conditions, we achieved up to 0.15%–0.3% reprogramming efficiencies. Subsequently, transgene-free hiPSCs were obtained by Cre-mediated excision of the reprogramming factors. The derived iPSCs maintained long-term self-renewal, normal karyotype and pluripotency, as demonstrated by the expression of stem cell markers and ability to form derivatives of three germ layers both in vitro and in vivo . Importantly, we demonstrated that Parkinson's patient transgene-free iPSCs derived using the same system could be directed towards differentiation into dopaminergic neurons under xeno-free culture conditions. Our approach provides a safe and robust platform for the generation of patient-specific iPSCs and derivatives for clinical and translational applications.